Department of Gastroenterology and Hepatology, University Hospital Coventry and Warwickshire NHS Trust, Clifford Bridge Road, Coventry CV2 2DX, UK; Warwick Medical School, University of Warwick, Coventry CV4 7HL, UK.
Mosaiques-Diagnostics GmbH, Hannover, Germany.
EBioMedicine. 2020 Dec;62:103083. doi: 10.1016/j.ebiom.2020.103083. Epub 2020 Nov 5.
Liver fibrosis is a consequence of chronic inflammation and is associated with protein changes within the hepatocytes structure. In this study, we aimed to investigate if this is reflected by the urinary proteome and can be explored to diagnose liver fibrosis in patients with chronic liver disease.
In a multicentre combined cross-sectional and prospective diagnostic test validation study, 129 patients with varying degrees of liver fibrosis and 223 controls without liver fibrosis were recruited. Additionally, 41 patients with no liver, but kidney fibrosis were included to evaluate interference with expressions of kidney fibrosis. Urinary low molecular weight proteome was analysed by capillary electrophoresis coupled to mass spectrometry (CE-MS) and a support vector machine marker model was established by integration of peptide markers for liver fibrosis.
CE-MS enabled identification of 50 urinary peptides associated with liver fibrosis. When combined into a classifier, LivFib-50, it separated patients with liver fibrosis (N = 31) from non-liver disease controls (N = 123) in cross-sectional diagnostic phase II evaluation with an area under the curve (AUC) of 0.94 (95% confidence intervals (CI): 0.89-0.97, p<0.0001). When adjusted for age, LivFib-50 demonstrated an AUC of 0.94 (95% CI: 0.89-0.97, p<0.0001) in chronic liver disease patients with (N = 19) or without (N = 17) liver fibrosis progression. In this prospective diagnostic phase III validation set, age-adjusted LivFib-50 showed 84.2% sensitivity (95% CI: 60.4-96.6) and 82.4% specificity (95% CI: 56.6-96.2) for detection of liver fibrosis. The sequence-identified peptides are mainly fragments of collagen chains, uromodulin and Na/K-transporting ATPase subunit γ. We also identified ten putative proteolytic cleavage sites, eight were specific for matrix metallopeptidases and two for cathepsins.
In liver fibrosis, urinary peptides profiling offers potential diagnostic markers and leads to discovery of proteolytic sites that could be targets for developing anti-fibrotic therapy.
肝纤维化是慢性炎症的后果，与肝细胞结构内的蛋白质变化有关。在这项研究中，我们旨在研究这是否反映在尿蛋白质组中，并可用于诊断慢性肝病患者的肝纤维化。
在一项多中心联合横断面和前瞻性诊断测试验证研究中，招募了 129 名不同程度肝纤维化的患者和 223 名无肝纤维化的对照组。此外，还纳入了 41 名无肝但有肾纤维化的患者，以评估肾纤维化表达的干扰。通过毛细管电泳与质谱（CE-MS）分析尿低分子量蛋白质组，并通过整合肝纤维化的肽标记物建立支持向量机标记模型。
CE-MS 能够鉴定出与肝纤维化相关的 50 种尿肽。当组合成一个分类器 LivFib-50 时，它在横断面诊断二期评估中，将肝纤维化患者（N=31）与非肝病对照组（N=123）分开，曲线下面积（AUC）为 0.94（95%置信区间（CI）：0.89-0.97，p<0.0001）。当调整年龄因素时，LivFib-50 在有（N=19）或无（N=17）肝纤维化进展的慢性肝病患者中，AUC 为 0.94（95%CI：0.89-0.97，p<0.0001）。在这项前瞻性诊断三期验证中，年龄调整后的 LivFib-50 对肝纤维化的检测显示出 84.2%的灵敏度（95%CI：60.4-96.6）和 82.4%的特异性（95%CI：56.6-96.2）。鉴定出的序列肽主要是胶原链、尿调素和 Na/K 转运 ATP 酶亚基 γ 的片段。我们还鉴定了十个潜在的蛋白水解切割位点，其中八个是基质金属蛋白酶特异性的，两个是组织蛋白酶特异性的。
在肝纤维化中，尿肽谱分析提供了潜在的诊断标志物，并发现了可能成为抗纤维化治疗靶点的蛋白水解位点。
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